The purpose of this work is to determine the molecular genetic changes that occur when one species splits into two species. Our long-term goals are to find genes that cause inviability in species hybrids, to determine their functions, and to infer their evolutionary histories. We use genetic tools from the model organism, Drosophila melanogaster, to perform mapping experiments in hybrids with the closely related species, D. simulans. In particular, the large collections of lethal chromosomal deletions and loss-of-function mutations from D. melanogaster are used to map hybrid inviability genes by complementation in F1 hybrid males. In previous work, 20 small regions of the D. simulans autosomal genome were identified that cause hybrid inviability via incompatible interactions with the genes on the D. melanogaster X chromosome. Here we propose to use new deletion-generating technology in D. melanogaster to make targeted, overlapping deletions in these regions that will allow us to narrow the cause hybrid inviability to single genes. We will use transgene experiments to confirm the identity of autosomal hybrid inviability genes and to identify their incompatible X-linked interactors. Once hybrid inviability genes are identified, we will study DNA sequence evolution to make inferences about the population genetic forces that drove their functional differentiation between species. This work will contribute to our understanding of how new animal species, including humans, evolve as a by-product of genetic divergence. Since we are concentrating on hybrid inviability, some of our work will involve characterizing previously unstudied viability-essential genes and gene interactions that have functions required in all eukaryotes, including humans.